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Cyclic ADP-ribose (cADPR) is an intracellular calcium mobilizer generated from NAD؉ by the ADP-ribosyl cyclases CD38 and BST-1. cADPR, both exogenously added and paracrinally produced by a CD38؉ feeder layer, has recently been demonstrated to stimulate the in vitro proliferation of human hemopoietic progenitors (HP) and also the in vivo expansion of hemopoietic stem cells. The low density of BST-1 expression on bone marrow (BM) stromal cells and the low specific activity of the enzyme made it unclear whether cADPR generation by a BST-1 ؉ stroma could stimulate HP proliferation in the BM microenvironment. We developed and characterized two BST-1 ؉ stromal cell lines, expressing an ectocellular cyclase activity similar to that of BST-1 ؉ human mesenchymal stem cells, the precursors of BM stromal cells. Long term co-culture of cord blood-derived HP over these BST-1 ؉ feeders determined their expansion. Influx of paracrinally generated cADPR into clonogenic HP was mediated by a concentrative, nitrobenzylthioinosine-and dipyridamole-inhibitable nucleoside transporter, this providing a possible explanation to the effectiveness of the hormone-like concentrations of the cyclic nucleotide measured in the medium conditioned by BST-1 ؉ feeders. These results suggest that the BST-1-catalyzed generation of extracellular cADPR, followed by the concentrative uptake of the cyclic nucleotide by HP, may be physiologically relevant in normal hemopoiesis.The self-renewal capacity of hemopoietic progenitors (HP) 1 in the bone marrow (BM) is a fundamental process in the physiology of hemopoiesis. The role of the BM stroma in providing HP with soluble factors essential to their proliferation and differentiation is well established (1, 2). However, the nature of the signals and the mechanisms by which stromal cells regulate the behavior of HP remain largely to be defined. Particularly, it is unknown whether perturbations of the balance between growth-stimulatory and -inhibitory factors may influence the expansion and self-renewal capacity of the hemopoietic reservoir.Recently, we have demonstrated that the potent intracellular calcium mobilizer cyclic ADP-ribose (cADPR) (3, 4) features properties of a novel hemopoietic growth factor (5, 6). Coinfusion of human HP with murine stromal cells transfected with the human ectocellular ADP-ribosyl cyclase CD38 improves hemopoietic stem cells engraftment into NOD/SCID mice (7). The paracrine interaction between stroma and HP has been investigated in a transwell co-culture setting where human HP were cultured over confluent monolayers of murine stromal cells transfected with human CD38: NAD ϩ efflux from stromal cells through connexin 43-formed hemichannels (8) provides CD38 with its substrate for ectocellular production of cADPR; subsequent influx of the cyclic nucleotide into HP (both committed and uncommitted progenitors) induces intracellular calcium ([Ca 2ϩ ] i ) mobilization and cell proliferation (6). In a similar co-culture setting, influx of cADPR into target 3T3 fibroblasts and dimet...

Lipopolysaccharide, the main component of the cell wall of Gram-negative bacteria, is known to activate microglial cells following its interaction with the CD14/Toll-like receptor complex (TLR-4). The activation pathway triggered by lipopolysaccharide in microglia involves enhanced basal levels of intracellular calcium ([Ca 2+ ] i ) and terminates with increased generation of cytokines/chemokines and nitric oxide. Here we demonstrate that in lipopolysaccharide-stimulated murine N9 microglial cells, cyclic ADP-ribose, a universal and potent Ca 2+ mobiliser generated from NAD + by ADP-ribosyl cyclases (ADPRC), behaves as a second messenger in the cell activation pathway. Lipopolysaccharide induced phosphorylation, mediated by multiple protein kinases, of the mammalian ADPRC CD38, which resulted in significantly enhanced ADPRC activity and in a 1.7-fold increase in the concentration of intracellular cyclic ADP-ribose. This event was paralleled by doubling of the basal [Ca 2+ ] i levels, which was largely prevented by the cyclic ADP-ribose antagonists 8-Br-cyclic ADPribose and ryanodine (by 75% and 88%, respectively). Both antagonists inhibited, although incompletely, functional events downstream of the lipopolysaccharide-induced microglia-activating pathway, i.e. expression of inducible nitric oxide synthase, overproduction and release of nitric oxide and of tumor necrosis factor a. The identification of cyclic ADP-ribose as a key signal metabolite in the complex cascade of events triggered by lipopolysaccharide and eventually leading to enhanced generation of pro-inflammatory molecules may suggest a new therapeutic target for treatment of neurodegenerative diseases related to microglia activation.

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